Rett syndrome (RTT) is a severe neurodevelopmental disorder that is caused by mutations in MeCP2, a transcriptional repressor that binds to methylated DNA. It is unclear how MeCP2 mutations lead to dysfunction of the nervous system in RTT, and no effective treatments for RTT are available. The overall goal of the present proposal is to characterize the properties of human neurons derived by in vitro differentiation of human embryonic stem cells (hESCs), and to elucidate the cellular and physiological impairments that are caused by the loss of MeCP2 in human neurons. We propose to utilize different NIH-registered hESC lines to produce human neurons, and to employ a small-hairpin RNA (shRNA) knockdown strategy to generate neurons that are deficient in MeCP2 and that can be used to study the effect of MeCP2 deficiency on neuronal gene expression and basic neuronal characteristics, such as morphogenesis, synaptogenesis, electrical properties, and synaptic function. The overall goal of the proposal is to determine the molecular, cellular and physiological impairments that are caused by MeCP2 deficiency in human neurons. Three Specific Aims are proposed: Specific Aim 1 will employ HSF1 and HSF6 hESC lines to produce human neural progenitor cells (hNPCs) and neurons, and to use shRNA knockdown and lentivirus infection to generate hNPCs and neurons that are deficient in MeCP2 or that express mutant forms of MeCP2 found in RTT. Specific Aim 2 will establish the effects of MeCP2 deficiency on fundamental neuronal characteristics, such as morphogenesis, synaptogenesis, electrical properties and synaptic function. Specific Aim 3 will analyze gene expression and epigenetic status (e.g., DNA methylation, histone modifications and miRNA expression) of MeCP2 deficient human neurons, and will identify MeCP2 target genes using ChIP-on-chip analysis. Through this approach, we will begin to link MeCP2 deficiency to alterations in the expression of direct MeCP2 target genes and their downstream genes, both of which may be associated with the neuronal phenotype. Overall, these experiments will provide initial insights into the function of MeCP2 in human neurons compared to rodent neurons. If fundamental differences in the effect of the MeCP2 decrease are observed between mouse and human neurons, our approach will allow analysis of the basis for these differences. If no such differences are observed, conversely, our data will provide a rationale for a wider use of mouse mutants for studying RTT. Collectively, the results of the proposed experiments will not only develop new disease models for RTT, but also help revealing the mechanisms of RTT using human neurons with the potential of identifying therapeutic targets. PUBLIC HEALTH RELEVANCE: Rett syndrome is a debilitating developmental brain disorder caused by mutations in a gene called MeCP2. In the brain, MeCP2 is known to regulate expression of genes, but how MeCP2 mutations impair the brain in Rett syndrome is unclear, and no effective treatments are available. We propose here to use human embryonic stem cells to generate human brain cells with a deficiency in MeCP2, and to study the effect of this deficiency on the development and functions of these brain cells, with the hope that the results will aid a better understanding of Rett syndrome, and facilitate the development of therapies for Rett syndrome.